Enzymatic oxidation of alcohols to aldehydes in a continuous reaction system

ABSTRACT

A process for the enzymatic oxidation of alcohols to aldehydes in a continuous reaction series is disclosed utilizing crude cellular methanol oxidase and catalase produced by an acceptable organism such as those selected from the genera of Pichia, Hansenula, Candida and Torulopsis. The process maintains a steady-state aldehyde concentration whereby enzyme activity is promoted. In particular, the process has two reactors operating in series continuously producing a fermenter effluent containing growing, intact yeast cells at a cell concentration of about 0.5 to about 3% by weight of the effluent by continuous culture of the cells on a methanol carbon source, continuously introducing the effluent from the first reactor to the second reactor, adding an alcohol feed at about 0.2-10% w/w and oxygen to the second reactor, maintaining a steady-state aldehyde concentration in the reaction mixture, the concentration of residual alcohol being about 0.1-9% w/w by controlling the rates of introduction of effluent and alcohol feed.

RELATED PATENT APPLICATION

The present invention is a continuation-in-part of U.S. patentapplication Ser. No 08/524,911 filed Sep. 8, 1995 now U.S. Pat. No.5,593,872 entitled "Enzymatic Oxidation of Alcohols to Aldehydes in aContinuous Reaction System Using Candida boidinii", which is acontinuation of U.S. patent application Ser. No. 08/165,748 filed Dec.10, 1993 entitled "Enzymatic Oxidation of Alcohols to Aldehydes in aContinuous Reaction System", now abandoned.

FIELD OF THE INVENTION

This invention relates generally to the production of aldehydes fromalcohols. More particularly, this invention relates to a process for theproduction of aldehydes in a continuous reaction by the enzymaticoxidation of alcohols utilizing crude alcohol oxidase and catalaseproduced by the yeast Candida boidinii or other acceptable organismssuch as those selected from the genera of Pichia, Hansenula, Torulopsisand other organisms selected from the genus Candida.

BACKGROUND OF THE INVENTION

Aldehydes are important chemicals in the food industry. They serve asnatural flavor additives to many food products. A preference amongconsumers for natural flavors exists as people are becoming moreconscientious about their health and diet. Acetaldehyde is especiallyutilized by the food manufacturers as it provides a fresh flavor to manyand various food products such as meats and breads. Acetaldehyde isimportant also for the synthesis of other desired products such asacetic acid and butanol.

Several methanol-utilizing microorganisms are capable of producingalcohol oxidase. These include yeasts of the genera Pichia, Hansenula,Candida, and Torulopsis, just to name a few. This alcohol oxidase canthen be used in the process of oxidizing alcohols such as ethanol intoaldehydes such as acetaldehyde.

U.S. Pat. No. 4,481,292 was issued for the production of acetaldehydefrom ethanol using an enzyme complex containing alcohol dehydrogenase,NADH, flavin mononucleotide, and a catalase. The '292 process requiresintricate steps of purification, separation and removal of components ofthe enzyme complex and products. This requires increased operationalcomplexity and increased time, thus reducing the economic feasibility.

U.S. Pat. No. 4,920,055 was issued for the production of an aldehyde andhydrogen peroxide from alcohols having five or fewer carbon atoms,utilizing a methanol oxidase produced by Hansenula polymorpha. The '055process uses a cell extract which is costly and results in yield loss.All examples in the '055 patent utilize purified enzymes. The process ofpurification reduces the economic feasibility of the process, as ittakes time and money to purify the extract. Another disadvantage of the'055 process is that the enzyme is maintained in the system by eitherimmobilization or recycling by use of membranes. Maintenance of thesufficient enzyme activity tends to be difficult by such immobilizationand/or recycling.

SUMMARY OF THE INVENTION

This invention provides an economically feasible process for thecontinuous oxidation of alcohols to aldehydes. Crude enzymes produced bymethanol-using organisms are used to oxidize the alcohols. Enzymes arecontinuously produced to oxidize the alcohols, thus maintainingsufficient enzyme activity.

The method of this invention involves enzymatically oxidizing alcoholsto aldehydes in a continuous reaction system. The reaction system hastwo reactors operated in series. In a first reactor an organism, such asCandida boidinii, produces methanol oxidase and catalase enzymes. Theseenzymes are then removed without purification from the first reactor andcombined with an aliphatic alcohol or aryl substituted aliphatic alcoholand oxygen in a second reactor, forming a reaction mixture. The alcoholin the second reactor is oxidized to its aldehyde. A steady-statealdehyde concentration in the reaction mixture of the second reactor ismaintained. The enzyme activity in the second reactor is promoted, whileother side reactions are eliminated. The aldehyde from the reactionmixture in the second reactor is then recovered.

In other subsidiary aspects of the invention, methanol oxidase oxidizesprimary alcohols having 2 to 7 carbons. These alcohols include arylsubstituted aliphatic alcohols such as benzyl alcohol, as well asaliphatic alcohols such as ethanol, n-propanol, n-butanol,2-chloroethanol, 2-bromoethanol, allyl alcohol, 2-buten-1-ol andmixtures thereof. In another aspect of the invention, the catalasereduces hydrogen peroxide in the second reactor to release half of theoxygen required for the oxidation of the primary alcohol. Air ispreferably added to reactor 2 and additional hydrogen peroxide may alsobe added. This additional hydrogen peroxide will be reduced by catalasealready present, and will provide the additional oxygen required for thereaction. This invention overcomes disadvantages associated with priorprocesses of the type maintained in the background above.

Various organisms may be used to produce methanol oxidase and catalaseenzymes such as Pichia pastoris, Pichia pinus, Pichia trehalophila,Pichia molischiana, Hansenula polymorpha, yeasts of Torulopsis andCandida, and all those yeasts which are genetically and/or taxonomicallyclosely related to Pichia, Hansenula, Torulopsis and Candida genera.

Additional objects, advantages and novel features of the invention willbe set forth in part in the description which follows, and others willbecome apparent to those skilled in the art upon examination of thisdescription or may be learned by practice of the invention. The objectsand advantages of the invention may be realized and attained by means ofthe instrumentalities and combinations particularly pointed out in theappended claims. The preferred embodiment is shown and described simplyby way of illustration of the best mode contemplated of carrying outthis invention. Accordingly, the FIGURE and description are to beregarded as illustrative in nature and not as restrictive.

BRIEF DESCRIPTION OF THE FIGURE

The FIGURE is a diagram of a continuous two-reactor system for oxidizingan alcohol to an aldehyde according to this invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENT

The preferred process of oxidizing alcohols to aldehydes uses methanoloxidase produced by certain microorganisms such as yeasts of the generaPichia, Candida, Hansenula and Torulopsis, and those which are closelyrelated to these genera. Other suitable yeasts may, however, be usedalso. The process of the present invention involves the growth of amethanol-utilizing organism such as Candida boidinii on methanol at acell density or concentration of about 0.5 to about 3% in the firstreactor to produce methanol oxidase and catalase. Methanol is used as acarbon source for the microorganism. The microorganism is grown incontinuous culture on the methanol medium. This medium is introduced tothe fermenter in the first reactor where oxidase and catalase aregenerated by the organism.

The fermenter effluent containing intra-cellular methanol oxidase andcatalase is introduced into the second reactor without purification. Amajor advantage of this invention is that extraction of the enzymes fromthe cells is not necessary. Also introduced into the second reactor isan aliphatic alcohol, or aryl substituted aliphatic alcohol containing 2to 7 carbons so that a reaction mixture is formed, particularly in aconcentration of about 0.2 to about 10% by weight feed alcohol. Air ispreferably continuously pumped into the second reactor and the methanoloxidase oxidizes the alcohol into the aldehyde and hydrogen peroxide.The hydrogen peroxide is then reduced to water and oxygen. As crudecellular enzymes in the fermenter effluent from the first reactor arecontinually being added to the second reactor, the enzymes in the secondreactor are replenished and the necessary enzyme activity is maintained.Therefore, the enzymes oxidize the alcohol in the second reactorproducing its aldehyde. The catalase in the second reactor reduceshydrogen peroxide to produce hydrogen and water. This removes thehydrogen peroxide from the reaction mixture, eliminating any unwantedside reactions, and providing part of the oxygen for the oxidationreaction. The residual alcohol concentration in the second reactor ismaintained particularly about 0.1 to about 9% by weight of the reactionmixture. The process of the invention, using crude alcohol oxidase andcatalase produced by a microorganism, thus oxidizes alcohols toaldehydes in an economically feasible manner.

EXAMPLE 1

The combinations and interactions of the features described above can bemore easily illustrated with reference to the schematic diagram of acontinuous reaction system shown in the FIGURE.

In the first reactor 1, having a working volume of 10 liters, aconcentration of about 2% of Candida boidinii is grown on a methanolmedium in continuous culture. The medium contains the following perliter of deionized water; 3.14 ml (86% strength) phosphoric acid; 2.12 gpotassium hydroxide pellets; 2.11 g MgSO₄.7H2O; 0.098 g CaCl₂.2H₂ O;0.068 g NaCl; 0.47 ml (96% strength) H₂ SO₄ ; 1.10 ml ammonium hydroxide(29% as NH₃); 1.5 mg biotin; 58 g methanol; and 16.3 ml micronutrientsolution. The micronutrient solution is composed of the following perliter of deionized water: 1.89 g FeSO4.7H₂ O; 0.99 g ZnSo₄.7H₂ O; 0.471g NaMo₄ ; 0.09 g KI; 0.075 g MnSO₄.H₂ O; 0.06 g CoCl₂.6H₂ O; 0.06 gCuSO₄.5H₂ O; and 5.5 g citric acid.

A 0.2u sterilizing filter 3 sterilizes the methanol medium as it passesthrough an outlet 12 and through the filter 3 at a volumetric dilutionrate of 0.08/hr. The growth medium ferments in the stirred reactor 1,with a pH maintained at about 2.5 to about 8, normally about 3.5, byautomatic addition of concentrated ammonium hydroxide 5 through an inlet6. The stirring speed of this medium is 850 rpm. The temperature ismaintained at about 20° C. to about 35° C., normally about 30° C., whilethe pressure is maintained at about 0 to about 3 atmospheres, normallyabout 6 psig. Air 8 is sparged into the first reactor 1 by way of aninlet 9 at a rate of 20 slpm. Foam is controlled by a mechanical foambreaker (not shown). Fermented effluent contains 0.1 unit/ml to about 20units/ml, normally about 10 to about 13 units/ml oxidase, about 0.02% ofresidual methanol, and about 20 g/l of cell dry weight.

The fermented effluent from the first reactor 1 is pumped withoutpurification through an outlet 11 into a stirred reactor 2. The secondreactor 2 has a 10 liter working volume. The pH of the second reactor 2is maintained at about 6 to about 8, normally about 7.2, by the additionof 50% w/w aqueous potassium hydroxide 13 through an inlet 14. About 92%ethanol 15 is added at about 3.6% by weight of the incoming fermentedeffluent through an inlet 16. The stirrer speed for this reactionmixture is 1,000 rpm. The temperature is maintained at about 5° C. toabout 37° C., normally about 20° C. It should be understood that thelower the temperature, the higher the concentration of aldehydeproduced. However, the oxidizing process is slow with lowertemperatures. The higher the temperature, the lower the concentration ofaldehyde produced. However, the oxidizing process is faster with highertemperatures. The pressure in the second reactor 2 is maintained atabout 0 atmospheres to about 3 atmospheres, normally atmospheric. Air 18is sparged into the second reactor 2 by way of an inlet 19, at a rate ofabout 0.1 to about 2 volumes air/volume liquid/min, normally about 0.3vvm. Foam is controlled by addition of an anti-foam 20 through an inlet21.

The reaction mixture maintains a steady state containing: about 1.3% w/wliquid acetaldehyde; about 2.67% w/w gaseous acetaldehyde; about 1.8%w/w residual ethanol; about 0.25% w/w gaseous ethanol. The acetaldehydeproductivity is about 1.72 g/l-h.

EXAMPLE 2

The same reaction process steps, reactants and reaction conditions areemployed as in Example 1 except that the yeast Pichia pastoris is grownon a methanol medium in continuous culture.

As like Example 1, the reaction mixture maintains a steady statecontaining: about 1.3% w/w liquid acetaldehyde; about 2.67% w/w gaseousacetaldehyde; about 1.8% w/w residual ethanol; about 0.25% w/w gaseousethanol. The acetaldehyde productivity is about 1.72 g/l-h.

EXAMPLE 3

The same reaction process steps, reactants and reaction conditions areemployed as in Example 1 except that the yeast Hansenula polymorpha isgrown on a methanol medium in continuous culture.

As like Example 1, the reaction mixture maintains a steady statecontaining: about 1.3% w/w liquid acetaldehyde; about 2.67% w/w gaseousacetaldehyde; about 1.8% w/w residual ethanol; about 0.025% w/w gaseousethanol. The acetaldehyde productivity is about 1.72 g/l-h.

EXAMPLE 4

The same reaction process steps, reactants and reaction conditions areemployed as in Example 1 except that the yeast is selected from thegenus Torulopsis and is grown on a methanol medium in continuousculture.

As like Example 1, the reaction mixture maintains a steady statecontaining: about 1.3% w/w liquid acetaldehyde; about 2.67% w/w gaseousacetaldehyde; about 1.8% w/w residual ethanol; about 0.25% w/w gaseousethanol. The acetaldehyde productivity is about 1.72 g/l-h.

Having described the invention, other embodiments will be understood toa person of ordinary skill in the art.

What is claimed is:
 1. A process for the enzymatic oxidation of alcoholsto aldehydes in a continuous reaction system of two reactors operated inseries comprising:continuously producing a fermenter effluent containinggrowing intact methylotrophic yeast cells at a cell concentration ofabout 0.5 to about 3% by weight of the effluent by continuous culture ofthe yeast cells using a carbon source consisting essentially of methanolin a first reactor, said yeast cells having intracellular methanoloxidase and catalase activity; continuously introducing the effluentfrom the first reactor without purification of the effluent, to a secondreactor; adding an alcohol feed and oxygen to the second reactor to formthe reaction mixture, said reaction mixture comprising about 0.2% toabout 10% by weight of said feed alcohol, the alcohol being selectedfrom the group consisting of ethanol, n-propanol, n-butanol,2-chloroethanol, 2-bromoethanol, allyl alcohol, 2-buten-1-ol, benzylalcohol and mixtures thereof; enzymatically oxidizing said alcohol inthe reaction mixture to its aldehyde while maintaining a steady-statealdehyde and alcohol concentration in the reaction mixture wherein saidalcohol concentration is about 0.1 to about 9% by weight of the reactionmixture by controlling the rates of introduction of said effluent andsaid alcohol feed to the reaction mixture; and recovering the aldehydefrom the reaction mixture.
 2. The process of claim 1 wherein said yeastcells are selected from the group consisting of the genera of Pichia,Hansenula, Torulopsis, and Candida.
 3. The process of claim 1 whereinsaid yeast cells are selected from the group consisting of Pichiapastoris, Pichia pinus, Pichia trehalophila, Pichia molischiana,Hansenula polymorpha, and Candida boidini.
 4. The process of claim 1wherein said temperature of the first reactor is maintained at about 20°C. to about 35° C., pressure is maintained at about 0 atmospheres toabout 3 atmospheres, and pH is maintained at about 2.5 to about
 8. 5.The process of claim 1 wherein said temperature of the second reactor ismaintained at about 5° C. to about 37° C., pressure is maintained atabout 0 atmospheres to about 3 atmospheres, and pH is maintained atabout 6 to about
 8. 6. The process of claim 1 wherein oxygen is suppliedby sparging air into the second reactor.
 7. The process of claim 6wherein said air is sparged at the rate of about 0.1 to about 2 volumeair/volume liquid/min.
 8. The process of claim 1 wherein oxygen issupplied by adding hydrogen peroxide to the second reactor which isreduced by said catalase.
 9. The process of claim 1 wherein said oxidaseconcentration in the first reactor is about 0.1 units/ml to about 20units/ml.